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de Vega M.,Institute Biologia Molecular Eladio Vinuela CSIC
PLoS ONE | Year: 2013

It is widely accepted that repair of double-strand breaks in bacteria that either sporulate or that undergo extended periods of stationary phase relies not only on homologous recombination but also on a minimal nonhomologous end joining (NHEJ) system consisting of a dedicated multifunctional ATP-dependent DNA Ligase D (LigD) and the DNA-end-binding protein Ku. Bacillus subtilis is one of the bacterial members with a NHEJ system that contributes to genome stability during the stationary phase and germination of spores, having been characterized exclusively in vivo. Here, the in vitro analysis of the functional properties of the purified B. subtilis LigD (BsuLigD) and Ku (BsuKu) proteins is presented. The results show that the essential biochemical signatures exhibited by BsuLigD agree with its proposed function in NHEJ: i) inherent polymerization activity showing preferential insertion of NMPs, ii) specific recognition of the phosphate group at the downstream 5′ end, iii) intrinsic ligase activity, iv) ability to promote realignments of the template and primer strands during elongation of mispaired 3′ ends, and v) it is recruited to DNA by BsuKu that stimulates the inherent polymerization and ligase activities of the enzyme allowing it to deal with and to hold different and unstable DNA realignments. © 2013 Miguel de Vega. Source


De Ory A.,Institute Biologia Molecular Eladio Vinuela CSIC | Zafra O.,Institute Biologia Molecular Eladio Vinuela CSIC | De Vega M.,Institute Biologia Molecular Eladio Vinuela CSIC
Nucleic Acids Research | Year: 2014

Intracellular reactive oxygen species as well as the exposure to harsh environmental conditions can cause, in the single chromosome of Bacillus subtilis spores, the formation of apurinic/apyrimidinic (AP) sites and strand breaks whose repair during outgrowth is crucial to guarantee cell viability. Whereas double-stranded breaks are mended by the nonhomologous end joining (NHEJ) system composed of an ATP-dependent DNA Ligase D (LigD) and the DNA-end-binding protein Ku, repair of AP sites would rely on an AP endonuclease or an AP-lyase, a polymerase and a ligase. Here we show that B. subtilis Ku (BsuKu), along with its pivotal role in allowing joining of two broken ends by B. subtilis LigD (BsuLigD), is endowed with an AP/deoxyribose 5′-phosphate (5′-dRP)-lyase activity that can act on ssDNA, nicked molecules and DNA molecules without ends, suggesting a potential role in BER during spore outgrowth. Coordination with BsuLigD makes possible the efficient joining of DNA ends with near terminal abasic sites. The role of this new enzymatic activity of Ku and its potential importance in the NHEJ pathway is discussed. The presence of an AP-lyase activity also in the homolog protein from the distantly related bacterium Pseudomonas aeruginosa allows us to expand our results to other bacterial Ku proteins. © 2014 The Author(s). Source


Perez-Arnaiz P.,Institute Biologia Molecular Eladio Vinuela CSIC | Lazaro J.M.,Institute Biologia Molecular Eladio Vinuela CSIC | Salas M.,Institute Biologia Molecular Eladio Vinuela CSIC | de Vega M.,Institute Biologia Molecular Eladio Vinuela CSIC
Journal of Molecular Biology | Year: 2010

DNA polymerases require two acidic residues to coordinate metal ions A and B at their polymerisation active site during catalysis of nucleotide incorporation. Crystallographic resolution of φ{symbol}29 DNA polymerase ternary complex showed that metal B coordination also depends on the carbonyl group of Val250 that belongs to the highly conserved Dx2SLYP motif of eukaryotic-type (family B) DNA polymerases. In addition, multiple sequence alignments have shown the specific conservation of this residue among the DNA polymerases that use a protein as primer. Thus, to ascertain its role in polymerisation, we have analysed the behaviour of single mutations introduced at the corresponding Val250 of φ{symbol}29 DNA polymerase. The differences in nucleotide binding affinity shown by mutants V250A and V250F with respect to the wild-type DNA polymerase agree to a role for Val250 as a metal B-dNTP complex ligand. In addition, mutant V250F was severely affected in φ{symbol}29 DNA replication because of a large reduction in the catalytic efficiency of the protein-primed reactions. In the light of the φ{symbol}29 DNA polymerase structures, a role for Val250 residue in the maintenance of the proper architecture of the enzyme to perform the protein-primed reactions is also proposed. © 2009 Elsevier Ltd. All rights reserved. Source

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